Two hypotheses currently exist regarding to the determining factors for sexual development and differentiation in birds. One is based on the unbalanced sex chromosome, meaning that avian sex determination is dominated by ¡§Z-chromosome dosage¡¨. The other brings up (reconsider this) the key factor of ¡§W chromosome¡¨ which is a particular sex chromosome in female birds (ZW). In the previous studies, we constructed a female-subtract-male cDNA library before morphological gonad differentiation. After sequencing and annotation, a total of 279 expression sequence taqs (ESTs) were identified, with potentially higher expression levels in females. By utilizing quantitative RT-PCR, 16 potential ESTs and three marker transcripts (HINT1, FET1 and WDR36), which identified to be involved in sexual development at 3, 5, 7, 9 days post-coitum (dpc) was analyzed in chicken embryos. Results indicated that AGR2, CPT2, DUSP19, HINTW, LOC771368 and EY53070791 had higher expression levels in female than in male embryos at 3 and 5 dpc; FET1 expression level in female embryos gradually increased from 3 to 9 dpc. Moreover, both HINT1 and WDR36 were higher expressed in male than in female embryos across 3 to 9 dpc. However, HINT1 exhibited higher expression levels starting at early stage, whereas WDR36 at later stage. Next, we constructed HINT1-GFP fusion protein and overexpressed this protein in chicken B-cell line (DT40), resulting in upregulation of WDR36 expression. On the contrary, overexpressed HINTW-GFP fusion protein in DT40 cells had decreased WDR36 expression level. Moreover, we designed a small hairpin RNA by utilizing RNA interference technique to knockdown expression of HINTW, which resulted in WDR36 upregulation. Finally, we then estimated the regulation of WDR36 promoter activity through analyzing HINT1-GFP overexpression. Results had shown that HINT1-GFP can improve WDR36 promoter activity. Therefore, we suppose that HINT1 can regulate WDR36 transcription via WDR36 proximal promoter region. Ongoing HINT1 responsive element(s) must be identified to characterize whether HINT1 or HINTW regulates WDR36.

From the sexual preselection point of view, understanding sex determination/differentiation mechanisms in the bird is critical in both evolutionary and industrial applications. The chicken embryo provides an unique vertebrate model in the field of development biology. Morphological sex development in the chick gonad starts at 6.5 embryonic day (E6.5), however, genetic sex determination and development should occur earlier. In order to comprehend genes and their underlying mechanisms being involved in sex-determination/development during early embryogenesis, we not only made a male-subtract-female and a female-subtract-male cDNA library as early as embryonic day 3 (E3; Hamburger and Hamilton Stage 20), but also examined early transcripts related to male development in chicken embryo and their expression profiles in this study. A total of 89 and 127 candidates of male-development transcripts represented respectively for 83 known and 119 unknown non-redundant sequences, which were characterized in an E3 male- subtract-female complementary DNA library. In this study, thirty-five selected transcripts being validated by quantitative reverse transcription-polymerase chain reaction that the expression levels of 25 transcripts were higher in male E3 whole embryos than in females (P < 0.05). Notably, twelve of these transcripts mapped to the Z chromosome. At 72 weeks of age, twenty transcripts were expressed at higher levels in testes than in ovaries. Meanwhile, four transcripts were expressed at higher levels in brains of male than in brains of female chickens (P < 0.05). By using of methods of whole mount and frozen cross-section in situ hybridization, the expressions of riboflavin kinase (RFK), WD repeat domain 36 (WDR36) and EY505808 transcripts on E7 chicken male gonads were corroborated to be better than female gonads. This result was confirmed by using of western blotting analysis which also showed the expressions were specifically on gonads than other tissues. Treatment with an aromatase inhibitor formestane at E4 depicted the effect of the expression levels at E7 of the coatomer protein complex (subunit beta 1), solute carrier family 35 member F1, LOC427316 and EY505812 transcripts across both sexes (P < 0.05), which was similar to the observed gene expressions for both doublesex and mab-3 related transcription factor 1 gene. Additionally, the interaction effects of sex with formestane treatment were observed in 15 candidate male development transcripts (P < 0.05). This study demonstrated a panel of potentially candidate male development transcripts being identified during early chicken embryogenesis; some might be regulated by sex hormones.